Detection method of ceftriaxone sodium polymer

Abstract

The invention belongs to the technical field of medicine quality control, and particularly relates to a detection method of a ceftriaxone sodium polymer. The invention discloses a polymer impurity needing to be controlled in a ceftriaxone sodium product, namely a ceftriaxone dimer D, and effectively improves the pertinence of polymer control in the ceftriaxone sodium product. On the basis, the method for detecting the ceftriaxone dimer D by the reversed-phase C18 high-performance liquid chromatography is developed, and the specificity, the quantitation limit, the detection limit, the precision, the linearity, the accuracy, the durability and the like of the detection method are verified. Results show that the detection method disclosed by the invention can accurately detect the ceftriaxone dimer D. Moreover, the invention also determines the forced polymerization condition of the ceftriaxone dimer D, the solution containing the forcibly polymerized ceftriaxone dimer D is used as the system adaptability solution of the detection method, the adaptability of the system is firstly verified during each detection, and a guarantee is provided for accurately detecting the impurities of the ceftriaxone dimer D.

Description

Technical Field

[0001] The invention belongs to the technical field of drug quality control, and particularly relates to a method for detecting a ceftriaxone sodium polymer. Background Art

[0002] β-lactam antibiotics are the most important class of antibiotics in clinical practice. The structure of this class of antibiotics is unstable, and the various organic impurities they contain are complex and unstable. Various types of polymerization between their molecules are prone to occur. Polymers are one of the key contents in the quality control research of this class of drugs. Polymer impurities are one of the important factors that cause allergic reactions to β-lactam antibiotics, but the generation mechanism of polymer impurities is complex and the pathways are diverse. The speed and intensity of the polymerization reaction of the product are affected by many factors. The types, quantities, and contents of polymers in the product are complex and diverse. The existing polymer control methods still have many inherent deficiencies in terms of detection specificity and accuracy.

[0003] Ceftriaxone sodium is a β-lactam antibiotic and one of the antibiotic products widely used in clinical practice. How to detect and control polymers is one of the key contents of the quality control research of this product. After inquiry, among the pharmacopoeias of major ICH member countries, only the Chinese Pharmacopoeia 2020 Edition Part II contains the ceftriaxone polymer detection method under the quality standard of ceftriaxone sodium. The ceftriaxone sodium polymer detection method included in the Chinese Pharmacopoeia 2020 Edition Part II uses molecular exclusion chromatography filtered by dextran gel G-10 to control the total amount of polymers in the product to not exceed 0.5%; however, this method has inherent defects, including low control accuracy, only the total amount of polymers can be detected, and the total amount of polymers detected may also contain other small molecule impurities, and the overall specificity of the detection method is not strong; at the same time, because the accuracy of this method cannot be verified, the precision and accuracy of the method cannot be determined.

[0004] Therefore, it is urgent to develop a detection method that can systematically verify the specificity, quantification limit, detection limit, precision, linearity, accuracy, durability and other technical indicators of polymer detection. Summary of the invention

[0005] In view of this, the present invention develops a method for detecting polymers that need to be controlled by using C18 reverse HPLC for polymers in ceftriaxone sodium and its preparations, and uses reference substances to systematically verify the specificity, quantitative limit, detection limit, precision, linearity, accuracy, durability, etc. of the method. The verification results show that the disclosed method can accurately and stably detect the polymer content in ceftriaxone sodium and its preparations.

[0006] In order to achieve the above object, the present invention adopts the following technical solution:

[0007] A method for detecting a ceftriaxone sodium polymer, wherein a C18 reverse high performance liquid chromatography method is used to detect a ceftriaxone sodium polymer that needs to be controlled, wherein the chemical name of the ceftriaxone sodium polymer is: (6R, 7R)-7-((Z)-2-(2-aminothiazol-4-yl)-2-(methoxyimino)acetylamino)-3-(((4-((Z)-2-(((6R, 7R)-2-carboxyl-3-(((2-methyl -5,6-dioxo-1,2,5,6-tetrahydro-1,2,4-triazine-3-yl)thio]methyl)-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-en-7-yl)amino)-1-(methoxyimino)-2-oxoethyl)thiazol-2-yl)amino)methyl)-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid; the structural formula is:

[0008]

[0009] It is worth noting that the existing polymer detection method is based on the molecular sieve theory. From a theoretical analysis, it detects the total amount of polymers, does not determine the type and structure of the polymer in the detected object, and cannot exclude whether the detected object contains other substances besides the polymer. Since the existing polymer detection method does not clearly define the type and structure of the detected object, it is impossible to verify whether the method is suitable for the specificity of polymer detection, the accuracy and precision of the detection. The present invention first clarifies the type and structure of the polymer that needs to be detected and controlled, and uses polymer impurity monomers to systematically verify the specificity, accuracy and precision of the method.

[0010] The present invention first uses a liquid chromatography-mass spectrometry method to detect multiple batches of ceftriaxone sodium preparations and reference preparations (ceftriaxone sodium for injection, trade name: Rocephin) samples. By analyzing the liquid chromatography-mass spectrometry test results, it can be seen that the polymer impurities mainly contained in ceftriaxone sodium are polymer (700), polymer (766-1), polymer (766-2), polymer (949-1), polymer (949-2), polymer (949-3) and polymer (967), and the specific structure is shown in Table 2. By comparing the compatible samples and the uncompatible samples, the types of polymer impurities detected in the compatible samples and the uncompatible samples are consistent. Before and after the compatibility, the content of individual polymers such as polymer (700) is within 0.05%, and the risk is relatively low, so there is no need to control it separately; while the content of polymer (949-2) in the compatible sample can increase from 0.02% before the compatibility to about 0.10%, and the content increase is the most obvious, and there is a risk of exceeding the quality control limit.

[0011] Therefore, in the present invention, the polymer (949-2) is named ceftriaxone dimer D, that is, the chemical name is (6R,7R)-7-((Z)-2-(2-aminothiazol-4-yl)-2-(methoxyimino)acetamido)-3-(((4-((Z)-2-(((6R,7R)-2-carboxy-3-(((2-methyl-5,6-dioxo-1,2,5,6-tetrahydro- Ceftriaxone sodium polymer of (1,2,4-triazine-3-yl)thio]methyl)-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-7-yl)amino)-1-(methoxyimino)-2-oxoethyl)thiazol-2-yl)amino)methyl)-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid. The polymer molecular formula is: C 32 H 31 N 13 O 12 S5, precise molecular weight is 949.08187.

[0012] Table 2

[0013]

[0014]

[0015] Further, the chromatographic conditions of C18 reverse phase HPLC are:

[0016] Octadecyl bonded silica was used as the filler; the column temperature was 28-32°C, the flow rate was 0.5-0.7 mL / min, and the detection wavelength was 268-272 nm;

[0017] 0.05 mol / L phosphate buffer was used as mobile phase A, and acetonitrile was used as mobile phase B;

[0018] The elution procedure is shown in Table 1:

[0019] Table 1

[0020]

[0021] Furthermore, the octadecyl bonded silica gel is a filler of C18, with a particle size of 3 μm, 4.6×250 mm, or a chromatographic column with equivalent performance;

[0022] The preparation method of the 0.05 mol / L phosphate buffer is as follows: 9.0 g of disodium hydrogen phosphate dodecahydrate and 3.5 g of anhydrous potassium dihydrogen phosphate are taken, dissolved in water and diluted to 1000 mL, and the pH is adjusted to 4.1-4.3 with phosphoric acid.

[0023] It is worth noting that, considering that ceftriaxone dimer D is a polar compound, the present invention uses reverse phase chromatography, and the chromatographic column uses octadecylsilane with weak polarity as a filling column. In order to make ceftriaxone dimer D emerge quickly, the pH value of the mobile phase is controlled at 4.1 to 4.3. A low concentration phosphate buffer system is used to maintain the stability of the pH value of the test system. By adjusting the organic phase ratio and the retention factor, ceftriaxone dimer D is separated from the interfering components.

[0024] Furthermore, the detection method of the C18 reverse HPLC method is:

[0025] 1) Take an appropriate amount of ceftriaxone sodium and obtain a system adaptability solution through pretreatment; take 20 μL of this solution and inject it into the chromatograph, record the chromatogram, and the separation degree between the ceftriaxone peak and the ceftriaxone sodium polymer peak and its previous adjacent peak should meet the requirements;

[0026] 2) Take an appropriate amount of ceftriaxone reference substance, add pH 7.0 phosphate buffer to dissolve and quantitatively dilute to make a 0.2 μg / mL solution, shake well as a sensitivity solution; take 20 μL of this solution and inject it into the chromatograph, record the chromatogram, and the signal-to-noise ratio of the ceftriaxone peak is not less than 10;

[0027] 3) Take an appropriate amount of ceftriaxone sodium, add pH 7.0 phosphate buffer to dissolve and quantitatively dilute to make a 1 mg / mL solution, shake well as the test solution; take 20 μL of this solution and inject it into the chromatograph, and record the chromatogram; accurately measure an appropriate amount of the test solution and add pH 7.0 phosphate buffer to dilute to make a 2 μg / ml solution, shake well as the control solution; take 20 μL of this solution and inject it into the chromatograph, and record the chromatogram.

[0028] It is worth noting that, in the present invention, the separation between the ceftriaxone peak and the ceftriaxone sodium polymer peak and their adjacent peaks is implemented in accordance with the general requirements in the Chinese Pharmacopoeia, and the "separation meets the requirements" is defaulted to "separation is not less than 1.5".

[0029] Furthermore, the relative retention time of the ceftriaxone sodium polymer is about 1.21, and the relative correction factor is about 1.2.

[0030] In the actual detection process of the product, the relative retention time will vary according to the filler modification functional groups of the chromatographic column, the filler particle size, etc. When there is no obvious interference before and after the target polymer peak, and the relative retention time of the chromatographic peak is about 1.21, the chromatographic peak can be determined as the target polymer peak; when there are multiple peaks that interfere with each other at the relative retention time of about 1.21, it is generally necessary to use a polymer impurity reference substance to further confirm the target polymer peak.

[0031] Furthermore, the preparation method of the pH 7.0 phosphate buffer is:

[0032] Take 6.805 g of anhydrous potassium dihydrogen phosphate and 0.94 g of sodium hydroxide, weigh accurately, add appropriate amount of water to dissolve, dilute with water to 1000 mL, adjust the pH value to 7.0 with 1 mol / L sodium hydroxide solution, and shake well to prepare a pH 7.0 phosphate buffer.

[0033] Furthermore, the pretreatment method for obtaining the system suitability solution through pretreatment in step 1) is:

[0034] Take an appropriate amount of ceftriaxone sodium, add pH 7.0 phosphate buffer to dissolve and dilute to a 5 mg / mL solution; accurately measure 1 mL of this solution, add 0.8-1.5 mL of 0.1 mol / L hydrochloric acid to destroy it for 0.5-1.2 hours, then add 0.1 mol / L sodium hydroxide solution to neutralize it, and add pH 7.0 phosphate buffer to dilute to a 0.25 mg / mL solution as the system adaptability solution;

[0035] Alternatively, take an appropriate amount of ceftriaxone sodium, add pH 7.0 phosphate buffer to dissolve and dilute to a 50 mg / mL solution; accurately measure 1 mL of this solution, place it in a transparent glass container, and then continuously destroy it under a light intensity of 5000±500Lx for 4±1 hours, and then add pH 7.0 phosphate buffer to dilute it to a 0.25 mg / mL solution as the system adaptability solution.

[0036] Compared with the prior art, the present invention discovers and discloses a structure of a polymer impurity that needs to be controlled in a ceftriaxone sodium product, namely ceftriaxone dimer D, which effectively improves the pertinence of polymer control in ceftriaxone sodium products. Based on this, a method for detecting ceftriaxone dimer D by reverse phase C18 high performance liquid chromatography was developed, and the specificity, quantitative limit, detection limit, precision, linearity, accuracy, durability, etc. of the detection method were verified. The results show that the detection method of ceftriaxone sodium polymer disclosed in the present invention can accurately detect ceftriaxone dimer D. In addition, the present invention also determines the forced polymerization conditions of ceftriaxone dimer D, and uses the ceftriaxone dimer D solution containing forced polymerization as the system adaptability solution of the above-mentioned detection method. The adaptability of the system is first verified each time the detection is performed, which provides a guarantee for accurately detecting ceftriaxone dimer D impurities. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying creative work.

[0038] Figure 1 It is a characteristic chromatogram under light destruction conditions in Example 1 of the present invention.

[0039] Figure 2 It is a characteristic chromatogram under alkali destruction conditions in Example 1 of the present invention.

[0040] Figure 3 It is a characteristic chromatogram under the conditions of oxidation destruction in Example 1 of the present invention.

[0041] Figure 4 It is a characteristic chromatogram under acid destruction conditions in Example 1 of the present invention.

[0042] Figure 5 It is a characteristic chromatogram of the concentrated solution under illumination conditions in Example 1 of the present invention.

[0043] Figure 6 This is the chromatogram of the solution not destroyed in Example 1 of the present invention.

[0044] Figure 7 It is a line graph of ceftriaxone in Example 2 of the present invention.

[0045] Figure 8 It is a line graph of ceftriaxone dimer D in Example 2 of the present invention. DETAILED DESCRIPTION

[0046] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0047] The word "embodiment" used here as an "exemplary" does not necessarily mean that any embodiment described is superior to or better than other embodiments. Unless otherwise specified, the performance index tests in the embodiments of this application are performed using conventional test methods in the art. It should be understood that the terms described in this application are only used to describe specific implementation methods and are not used to limit the content disclosed in this application.

[0048] Unless otherwise specified, the technical and scientific terms used in this document have the same meanings as commonly understood by ordinary technicians in the technical field to which this application belongs; other experimental methods and technical means not specifically specified in this application refer to experimental methods and technical means commonly used by ordinary technicians in this field.

[0049] In order to better illustrate the content of the present application, numerous specific details are provided in the specific examples below. It should be understood by those skilled in the art that the present application can also be implemented without certain specific details. In the embodiments, some methods, means, instruments, equipment, etc. well known to those skilled in the art are not described in detail in order to highlight the main purpose of the present application.

[0050] Under the premise of no conflict, the technical features disclosed in the embodiments of the present application can be combined arbitrarily, and the resulting technical solutions belong to the contents disclosed in the embodiments of the present application.

[0051] The present invention discovers and discloses a structure of a polymer impurity that needs to be controlled in a ceftriaxone sodium product, namely ceftriaxone dimer D, which effectively improves the pertinence of polymer control in ceftriaxone sodium products. Based on this, a method for detecting ceftriaxone dimer D by reverse phase C18 high performance liquid chromatography was developed, and the specificity, quantitative limit, detection limit, precision, linearity, accuracy, durability, etc. of the detection method were verified. The results show that the detection method of ceftriaxone sodium polymer disclosed in the present invention can accurately detect ceftriaxone dimer D. In addition, the present invention also determines the forced polymerization conditions of ceftriaxone dimer D, and uses the ceftriaxone dimer D solution containing forced polymerization as the system adaptability solution of the above-mentioned detection method. The adaptability of the system is first verified each time the detection is performed, which provides a guarantee for accurately detecting ceftriaxone dimer D impurities.

[0052] The detection method used in each embodiment of the present invention is:

[0053] Determined according to high performance liquid chromatography (Chinese Pharmacopoeia 2020 Edition, Part IV, General Rules 0512).

[0054] Chromatographic conditions:

[0055] Octadecyl bonded silica gel is used as filler (C18, particle size: 3μm, 4.6×250mm, or chromatographic column with equivalent performance); column temperature is 30℃, flow rate is about 0.6mL per minute, and detection wavelength is 270nm. 0.05mol / L phosphate buffer (take 9.0g disodium hydrogen phosphate dodecahydrate and 3.5g anhydrous potassium dihydrogen phosphate, dissolve in water and dilute to 1000mL, adjust pH to 4.2 with phosphoric acid) is used as mobile phase A, and acetonitrile is used as mobile phase B.

[0056] The elution procedure is shown in Table 1:

[0057] Table 1

[0058]

[0059] Take 6.805 g of anhydrous potassium dihydrogen phosphate and 0.94 g of sodium hydroxide, weigh accurately, add appropriate amount of water to dissolve, dilute to 1000 mL with water, adjust the pH value to 7.0 with 1 mol / L sodium hydroxide solution, and shake well to prepare pH 7.0 phosphate buffer.

[0060] Take an appropriate amount of ceftriaxone sodium, add pH 7.0 phosphate buffer to dissolve and dilute to a 5 mg / mL solution; accurately measure 1 mL of this solution, add 0.8 mL of 0.1 mol / L hydrochloric acid to destroy it for 1.2 hours, then add 0.1 mol / L sodium hydroxide solution to neutralize it, and add pH 7.0 phosphate buffer to dilute it to a 0.25 mg / mL solution as the system adaptability solution; take 20 μL of this solution and inject it into the chromatograph, record the chromatogram, and the separation degree between the ceftriaxone peak and the ceftriaxone sodium polymer peak and its previous adjacent peak should meet the requirements.

[0061] Take an appropriate amount of ceftriaxone reference substance, add pH 7.0 phosphate buffer to dissolve and quantitatively dilute to make a 0.2 μg / mL solution, shake well as a sensitivity solution; take 20 μL of this solution and inject it into the chromatograph, record the chromatogram, and the signal-to-noise ratio of the ceftriaxone peak is not less than 10.

[0062] The relative retention time of ceftriaxone dimer D was about 1.21, and the relative correction factor was about 1.2.

[0063] Take an appropriate amount of ceftriaxone sodium, add pH 7.0 phosphate buffer to dissolve and quantitatively dilute to make a 1 mg / ml solution, shake well as the test solution; take 20 μL of this solution and inject it into the chromatograph, and record the chromatogram; accurately measure an appropriate amount of the test solution and add pH 7.0 phosphate buffer to dilute to make a 2 μg / ml solution, shake well as the control solution; take 20 μL of this solution and inject it into the chromatograph, and record the chromatogram; if the ceftriaxone sodium polymer peak can be detected in the chromatogram of the test solution, the peak area of ​​the ceftriaxone sodium polymer peak shall not be greater than 1 times (0.2%) the peak area of ​​the main peak of the control solution.

[0064] In order to better understand the present invention, the present invention is further specifically described below through the following examples, but it should not be understood as a limitation of the present invention. Some non-essential improvements and adjustments made by technicians in this field based on the above invention content are also considered to fall within the protection scope of the present invention.

[0065] Example 1 System Applicability Solution Preparation Conditions

[0066] (1) Test sample stock solution: Accurately weigh 50 mg of ceftriaxone sodium and place it in a 10 mL volumetric flask. Add diluent to dissolve and dilute to the mark, and shake well.

[0067] (2) Undamaged solution: Accurately measure 1 mL of the stock solution of the test sample into a 5 mL volumetric flask, add diluent to dissolve and dilute to the mark, and shake well.

[0068] (3) Acid destruction: Accurately measure 1 mL of the sample stock solution into a 5 mL volumetric flask, add 0.8 mL of 0.1 mol / L hydrochloric acid solution, and leave at room temperature for 1 h. Add 1 mL of 0.1 mol / L sodium hydroxide solution to neutralize, add diluent to dissolve and dilute to the scale, and shake well.

[0069] (4) Alkali destruction solution: Accurately measure 1 mL of the sample stock solution into a 5 mL volumetric flask, add 1 mL of 0.1 mol / L sodium hydroxide solution, let stand at room temperature for 3 min, add 1 mL of 0.1 mol / L hydrochloric acid solution and diluent to dissolve and dilute to the scale, and shake well.

[0070] (5) Light damage: Accurately measure 1 mL of the sample stock solution into a 5 mL transparent centrifuge tube and place it in a drug stability test chamber (illuminance: 5000 ± 500 Lx) for 5 h. Add diluent to dissolve and dilute to the scale, and shake well.

[0071] (6) Oxidative damage: Accurately measure 1 mL of the sample stock solution into a 5 mL volumetric flask, add 1 mL of 0.03% hydrogen peroxide solution, leave at room temperature for 15 min, add diluent to dissolve and dilute to the mark, and shake well.

[0072] (7) Concentrated solution test sample stock solution: Accurately weigh 500 mg of ceftriaxone sodium, place it in a 10 mL volumetric flask, add diluent to dissolve and dilute to the mark, and shake well. Concentrated solution light damage: Accurately measure 1 mL of concentrated solution test sample stock solution and place it in a transparent centrifuge tube, place it in a drug stability test chamber (illuminance: 5000±500Lx) for 3 hours, then accurately measure 1 mL to a 50 mL volumetric flask, add diluent to dissolve and dilute to the mark, and shake well.

[0073] The obtained system suitability condition screening results are shown in Table 3.

[0074] Table 3

[0075]

[0076] The test sample solutions were taken and destroyed under different conditions. The results showed that ( Figure 1 to Figure 6), the content of ceftriaxone dimer impurity D did not change significantly when the sample was destroyed by light for 5h; the content of ceftriaxone dimer impurity D did not change when the sample was oxidized for 15min and destroyed by alkali for 3min; the content of ceftriaxone dimer impurity D increased significantly when the sample was destroyed by acid for 1h and when the sample concentrated solution was irradiated with light (illuminance: 5000±500Lx) for 3h, indicating that the ceftriaxone dimer impurity D increased significantly under acidic conditions and concentrated solution illumination conditions.

[0077] The structural characteristics of the above-mentioned ceftriaxone dimer impurity D are as follows:

[0078] In the aqueous solution of ceftriaxone sodium under acidic and light conditions, the CS bond formed by the C at position 18 and the S at position 19 is unstable and easily broken, and combines with the H2N at position 14 to polymerize into the ceftriaxone dimer impurity D. The generation process is as follows:

[0079]

[0080] Example 2

[0081] The ceftriaxone dimer D detection method disclosed in the present invention was systematically validated in terms of methodology, including specificity, limit of quantification, limit of detection, precision, accuracy, durability, etc. The validation results show that the selected method can accurately and stably detect the polymer content in the homemade preparation. The specific validation results are summarized as follows:

[0082] 2.1 Exclusivity

[0083] (1) Blank solvent: phosphate buffer, pH 7.0.

[0084] (2) Sensitivity solution: Accurately weigh 20 mg of ceftriaxone reference substance and place it in a 100 mL volumetric flask, add pH 7.0 phosphate buffer to dissolve and make up to volume, and shake well. Accurately measure 1.0 mL of the above solution and place it in a 100 mL volumetric flask, add pH 7.0 phosphate buffer to make up to volume, and shake well. Accurately measure 2.0 mL of this solution and place it in a 20 mL volumetric flask, add pH 7.0 phosphate buffer to make up to volume, and shake well.

[0085] (3) System adaptability solution: Accurately weigh 100 mg of ceftriaxone sodium injection preparation, place it in a 20 mL volumetric flask, add pH 7.0 phosphate buffer to dissolve and shake to volume. Accurately measure 1.0 mL of this solution and place it in a 20 mL volumetric flask, add 1.0 mL of 0.1 mol / L hydrochloric acid to disrupt it for 1 hour, then add 0.1 mol / L sodium hydroxide solution to neutralize it, and add pH 7.0 phosphate buffer to dilute and make up the volume.

[0086] (4) Ceftriaxone reference solution: Accurately weigh about 5 mg of ceftriaxone reference substance into a 25 mL volumetric flask, add pH 7.0 phosphate buffer to dissolve and dilute to the mark.

[0087] Trans-cefotaxime control solution: Accurately weigh 5.0 mg of trans-cefotaxime into a 20 mL volumetric flask, add pH 7.0 phosphate buffer to dissolve and dilute to the mark to obtain trans-cefotaxime sodium stock solution. Accurately weigh 1.0 mL of trans-cefotaxime sodium stock solution into a 25 mL volumetric flask, add pH 7.0 phosphate buffer to dilute to the mark, and shake well to obtain trans-cefotaxime control solution.

[0088] (5) Ceftriaxone Impurity B Reference Solution: Accurately weigh 2.5 mg of Ceftriaxone Impurity B into a 20 mL volumetric flask, add pH 7.0 phosphate buffer to dissolve and dilute to scale to obtain Ceftriaxone Impurity B stock solution. Accurately weigh 1.0 mL of Ceftriaxone Impurity B stock solution into a 25 mL volumetric flask, add pH 7.0 phosphate buffer to dilute to scale, and shake well to obtain Ceftriaxone Impurity B Reference Solution.

[0089] (6) Ceftriaxone Impurity C Reference Solution: Accurately weigh 5.0 mg of Ceftriaxone Impurity C into a 20 mL volumetric flask, add pH 7.0 phosphate buffer to dissolve and dilute to scale to obtain Ceftriaxone Impurity C stock solution. Accurately weigh 1.0 mL of Ceftriaxone Impurity C stock solution into a 25 mL volumetric flask, add pH 7.0 phosphate buffer to dilute to scale, and shake well to obtain Ceftriaxone Impurity C reference solution.

[0090] (7) Ceftriaxone Impurity D Reference Solution: Accurately weigh 1.0 mg of Ceftriaxone Impurity D into a 20 mL volumetric flask, add pH 7.0 phosphate buffer to dissolve and dilute to scale to obtain Ceftriaxone Impurity D stock solution. Accurately weigh 1.0 mL of Ceftriaxone Impurity D stock solution into a 25 mL volumetric flask, add pH 7.0 phosphate buffer to dilute to scale, and shake well to obtain Ceftriaxone Impurity D Reference Solution.

[0091] (8) Ceftriaxone Impurity E Reference Solution: Accurately weigh 1.0 mg of Ceftriaxone Impurity E into a 20 mL volumetric flask, add pH 7.0 phosphate buffer to dissolve and dilute to the mark to obtain Ceftriaxone Impurity E stock solution. Accurately weigh 1.0 mL of Ceftriaxone Impurity E stock solution into a 25 mL volumetric flask, add pH 7.0 phosphate buffer to dilute to the mark, and shake well to obtain Ceftriaxone Impurity E Reference Solution.

[0092] (9) Ceftriaxone Dimer D control solution: Accurately weigh 1 mg of Ceftriaxone Dimer D into a 20 mL volumetric flask, add pH 7.0 phosphate buffer to dissolve and dilute to scale as the Ceftriaxone Dimer D stock solution. Accurately weigh 1.0 mL of Ceftriaxone Dimer D stock solution into a 25 mL volumetric flask, add pH 7.0 phosphate buffer to make up to volume.

[0093] (10) Self-prepared test solution: Accurately weigh 25 mg of the self-prepared ceftriaxone sodium injection into a 25 mL volumetric flask, add pH 7.0 phosphate buffer to dissolve and dilute to the mark.

[0094] (11) Reference product test solution: Accurately weigh 25 mg of the reference product of ceftriaxone sodium for injection into a 25 mL volumetric flask, dissolve in pH 7.0 phosphate buffer and dilute to the mark.

[0095] (12) Spiked homemade preparation test solution: Accurately weigh 25 mg of homemade ceftriaxone sodium injection preparation into a 25 mL volumetric flask, add pH 7.0 phosphate buffer to dissolve, accurately add 1.0 mL of each impurity stock solution and then dilute to the mark with pH 7.0 phosphate buffer.

[0096] The experiment shows (Table 4) that the blank solvent has no interference. The separation degree of the ceftriaxone peak and its adjacent peak before it in the system suitability solution is 11.760, and the separation degree of the ceftriaxone peak and its adjacent peak after it is 8.574; the separation degree of the ceftriaxone dimer D peak and its adjacent peak before it is 7.751, and the separation degree of the ceftriaxone dimer D peak and its adjacent peak after it is 6.415. The main peak signal-to-noise ratio of the sensitivity solution is 21.8. The presence of each known impurity peak does not interfere with the detection of ceftriaxone dimer D. The peak area of ​​the ceftriaxone dimer peak in the spiked test solution is significantly increased.

[0097] Table 4 Specificity test results

[0098]

[0099] 2.2 Limit of quantification

[0100] Blank solvent: phosphate buffer, pH 7.0.

[0101] Take an appropriate amount of ceftriaxone reference substance, dissolve it in pH 7.0 phosphate buffer and dilute it step by step, then inject 20 μL of the resulting solution into the chromatograph. The concentration of ceftriaxone when the signal of the ceftriaxone peak on the recorded chromatogram is about 10 times the noise is taken as the quantitative limit under the chromatographic conditions, and the injection is repeated 6 times.

[0102] Take an appropriate amount of ceftriaxone dimer D reference substance, dissolve it in pH 7.0 phosphate buffer and dilute it step by step, then inject 20 μL of the resulting solution into the chromatograph. The concentration of ceftriaxone dimer D when the signal of the ceftriaxone dimer D peak on the recorded chromatogram is about 10 times the noise is taken as the quantitative limit under the chromatographic conditions, and the injection is repeated 6 times.

[0103] The control solution with a concentration of 0.174428 mg / mL of ceftriaxone was diluted 1000 times, and 20 μL of the diluted control solution was injected into the chromatograph. The result showed that the quantification limit of ceftriaxone was 174.43 ng / mL, and the quantification limit was 3.49 ng. The control solution with a concentration of 0.046064 mg / mL of ceftriaxone dimer D was diluted 125 times, and 20 μL of the diluted control solution was injected into the chromatograph. The result showed that the quantification limit of ceftriaxone dimer D was 368.51 ng / ml, and the quantification limit was 7.37 ng.

[0104] Table 5 Quantitation limit experimental results

[0105]

[0106] Accordingly, in the detection method of ceftriaxone sodium polymer disclosed in the present invention, the quantification limit of ceftriaxone is 3.49 ng, and the peak area RSD is 5.40%, indicating that the quantification limit solution of ceftriaxone has good repeatability; the quantification limit of ceftriaxone dimer D is 7.37 ng, and the peak area RSD is 1.04%, indicating that the quantification limit solution of ceftriaxone dimer D has good repeatability.

[0107] 2.3 Detection limit

[0108] Blank solvent: phosphate buffer, pH 7.0.

[0109] Take an appropriate amount of ceftriaxone reference substance, dissolve it in pH 7.0 phosphate buffer and dilute it step by step, then inject 20 μL of the resulting solution into the chromatograph. The concentration of ceftriaxone when the signal of the ceftriaxone peak on the recorded chromatogram is about 3 times the noise is taken as the detection limit under the chromatographic conditions, and repeat the injection 3 times.

[0110] Take an appropriate amount of ceftriaxone dimer D reference substance, dissolve it in pH 7.0 phosphate buffer and dilute it step by step, then inject 20 μL of the resulting solution into the chromatograph. The concentration of ceftriaxone dimer D when the signal of the ceftriaxone dimer D peak on the recorded chromatogram is about 3 times the noise is taken as the detection limit under the chromatographic conditions, and the injection is repeated 3 times.

[0111] A control solution with a concentration of 0.174428 mg / mL of ceftriaxone was diluted 4000 times, and 20 μL of the diluted control solution was injected into the chromatograph. The results showed that the detection limit of ceftriaxone was 43.61 ng / m and the detection limit was 0.87 ng. A control solution with a concentration of 0.046064 mg / mL of ceftriaxone dimer D was diluted 250 times, and 20 μL of the diluted control solution was injected into the chromatograph. The detection limit of ceftriaxone dimer D was 184.25 ng / ml, and the detection limit was 3.68 ng.

[0112] Table 6 Detection limit experimental results

[0113]

[0114] Accordingly, in the detection method of ceftriaxone sodium polymer disclosed in the present invention, the detection limit of ceftriaxone is 0.87 ng, and the peak area RSD is 11.91%, indicating that the detection limit solution of ceftriaxone has good repeatability; the detection limit of ceftriaxone dimer D is 3.68 ng, and the peak area RSD is 1.37%, indicating that the detection limit solution of ceftriaxone dimer D has good repeatability.

[0115] 2.4 Precision

[0116] (1) Injection precision

[0117] Accurately weigh 25 mg of the content of ceftriaxone sodium for injection into a 25 mL volumetric flask, add pH 7.0 phosphate buffer to dissolve and dilute to scale. Accurately measure 1.0 mL of this solution into a 100 mL volumetric flask, add pH 7.0 phosphate buffer to dilute to scale and shake well. Accurately measure 5.0 mL of this solution into a 25 mL volumetric flask, add pH 7.0 phosphate buffer to the volume, and shake well as a self-control solution. Take 20 μL of the resulting solution and inject it into the chromatograph, repeat the injection 6 times, record the chromatogram, and calculate the RSD value of the peak area.

[0118] Accurately weigh 1 mg of ceftriaxone dimer D into a 20 mL volumetric flask, add pH 7.0 phosphate buffer to dissolve and dilute to scale as the stock solution of ceftriaxone dimer D. Accurately measure 1.0 mL of ceftriaxone dimer D stock solution into a 25 mL volumetric flask, add mobile phase to dilute to scale and shake well as the reference solution of ceftriaxone dimer D. Take 20 μL of the resulting solution and inject it into the chromatograph, repeat the injection 6 times, record the chromatogram, and calculate the RSD value of the peak area.

[0119] Table 7 Injection precision experimental results

[0120]

[0121]

[0122] As can be seen from Table 7, the RSD value of the peak area of ​​ceftriaxone detected by 6 consecutive injections of the self-control solution is 0.55%, and the RSD value of the retention time is 0.11%; the RSD value of the peak area of ​​ceftriaxone dimer D detected by 6 consecutive injections of the ceftriaxone dimer D control solution is 0.62%, and the RSD value of the retention time is 0.085%, indicating that the detection method of ceftriaxone sodium polymer disclosed in the present invention has good injection precision.

[0123] (2) Repeatability

[0124] Take 6 portions of the test sample (cefotaxime sodium for injection, batch number: 20021322), about 25 mg each, accurately weigh, place in 25 mL volumetric flasks, add pH 7.0 phosphate buffer to dissolve and dilute to the scale, shake well as the test sample solution.

[0125] Accurately measure 1.0 mL of the test solution and place it in a 100 mL volumetric flask, add pH 7.0 phosphate buffer to dilute to the mark and shake well. Accurately measure 5.0 mL of this solution and place it in a 25 mL volumetric flask, add pH 7.0 phosphate buffer to the same volume, and shake well as the control solution.

[0126] Accurately pipette 20 μL of each test solution and control solution, inject them into the liquid chromatograph, and record the chromatogram.

[0127] Table 8 Repeatability test results

[0128]

[0129] As can be seen from Table 8, the average content of ceftriaxone dimer D in the 6 test samples was 0.02%, which was less than 0.03%. The results were not compared, indicating that the detection method of ceftriaxone sodium polymer disclosed in the present invention has good repeatability.

[0130] (3) Reproducibility

[0131] An experimenter from the R&D department and an experimenter from the quality inspection department tested the ceftriaxone dimer D content of the same batch of homogeneity samples (cefotaxime sodium for injection, batch number: 20021322) on different dates and in different laboratories according to the published method.

[0132] Take 6 portions of the test sample (cefotaxime sodium for injection, batch number: 20021322), about 25 mg each, accurately weigh, place in 25 mL volumetric flasks, add pH 7.0 phosphate buffer to dissolve and dilute to the scale, shake well as the test sample solution.

[0133] Accurately measure 1.0 mL of the test solution into a 100 mL volumetric flask, add pH 7.0 phosphate buffer to dilute to the mark and shake well. Accurately measure 5.0 mL of this solution into a 25 mL volumetric flask, add pH 7.0 phosphate buffer to the mark and shake well as the control solution.

[0134] Accurately pipette 20 μl of each test solution and control solution, inject them into the liquid chromatograph respectively, and record the chromatogram.

[0135] Table 9 Reproducibility test results

[0136]

[0137] It can be seen from Table 9 that the content of ceftriaxone dimer D in the 6 repeatable test solutions was less than 0.03%. The combined analysis results of the 6 repeatable test solutions showed that the content of ceftriaxone dimer D in the 12 test solutions was less than 0.03%, which met the acceptable standard, indicating that the method had good reproducibility.

[0138] 2.5 Accuracy

[0139] (1) Ceftriaxone accuracy

[0140] The concentration of the ceftriaxone control solution is set to the maximum amount of ceftriaxone dimer D contained in the test sample (the limit value of ceftriaxone dimer D is 0.2%), and the ceftriaxone content is proposed to be 40%, 100%, and 160%, respectively. Accurately weigh 25 mg of the ceftriaxone reference substance, add pH 7.0 phosphate buffer to dissolve and dilute to 25 mL. Accurately measure 1.0 mL of this solution to a 100 mL volumetric flask, add pH 7.0 phosphate buffer to dilute to the scale, and shake well to obtain the ceftriaxone stock solution. Accurately measure 2.0 mL, 5.0 mL, and 8.0 mL of ceftriaxone stock solution to a 25 mL volumetric flask and add pH 7.0 phosphate buffer to the volume as solutions with accuracy of 40%, 100%, and 160%.

[0141] Accurately weigh 25 mg of ceftriaxone reference substance, dissolve and dilute to 25 mL with pH 7.0 phosphate buffer. Accurately measure 1.0 mL of this solution into a 100 mL volumetric flask and dilute to scale with pH 7.0 phosphate buffer. Accurately measure 5.0 mL of this solution into a 25 mL volumetric flask and dilute to volume with pH 7.0 phosphate buffer as the ceftriaxone reference solution.

[0142] Take the above accuracy solution and reference solution, measure them separately, and calculate by peak area according to the external standard method.

[0143] Table 10 Ceftriaxone accuracy recovery experimental results

[0144]

[0145] The average recovery of ceftriaxone at each concentration was between 97.93% and 104.87%, the total average recovery was 100.64%, the RSD value was 3.1%, and the recovery of each sample met the acceptable standard, indicating that this method can accurately detect the content of ceftriaxone.

[0146] (2) Ceftriaxone Dimer D Accuracy

[0147] The content of ceftriaxone dimer D reference solution was set to the maximum amount in the quality standard (the limit value of ceftriaxone dimer D was 0.2%), and the proposed ceftriaxone dimer D contents were 25%, 100%, and 150%, respectively.

[0148] Accurately weigh 25 mg of ceftriaxone sodium for injection, place in a 25 mL volumetric flask, add pH 7.0 phosphate buffer to dissolve and dilute to the scale, and shake well as the test solution.

[0149] Accurately weigh 1 mg of ceftriaxone dimer D reference substance and place it in a 20 mL volumetric flask, add pH 7.0 phosphate buffer to dissolve and dilute to the scale, and shake well to obtain the stock solution of ceftriaxone dimer D. Take 1 mL of ceftriaxone dimer D stock solution and place it in a 25 mL volumetric flask, add pH 7.0 phosphate buffer to dissolve and dilute to volume as the reference substance solution.

[0150] Accurately weigh 100 mg of ceftriaxone sodium for injection, place in a 100 mL volumetric flask, add pH 7.0 phosphate buffer to dissolve, then add 1 mL, 4 mL, and 6 mL of ceftriaxone dimer D stock solution respectively, then add pH 7.0 phosphate buffer to dissolve, dilute and fix volume to prepare solutions with accuracy of 25%, 100%, and 150%.

[0151] Take the above-mentioned reference solution, test solution and accuracy solution, measure them separately, and calculate the peak area according to the external standard method.

[0152] Table 11 Ceftriaxone Dimer D Accuracy Recovery Experimental Results

[0153]

[0154] It can be seen from Table 11 that the average recovery rate of ceftriaxone dimer D at each concentration is between 98.23% and 103.04%, the total average recovery rate is 100.70%, the RSD value is 1.7%, and the recovery rate of each sample meets the acceptable standard, indicating that this method can accurately detect the content of ceftriaxone dimer D.

[0155] 2.6 Linearity

[0156] (1) Ceftriaxone linear

[0157] Accurately weigh 25.66 mg of ceftriaxone reference substance, dissolve and dilute to 25 mL with pH 7.0 phosphate buffer. Accurately measure 1 mL of this solution and place it in a 100 mL volumetric flask, add pH 7.0 phosphate buffer to make up to the volume to obtain a reference substance stock solution with a ceftriaxone concentration of 0.008611 mg / mL. Accurately measure an appropriate amount of the reference substance stock solution and dilute it with pH 7.0 phosphate buffer to prepare a series of reference substance solutions with concentrations of 0.000172 mg / mL, 0.000344 mg / mL, 0.001033 mg / mL, 0.001722 mg / mL, 0.002583 mg / mL, and 0.003445 mg / mL, respectively. Accurately aspirate 20 μL of each series of reference substance solutions, inject them into the chromatograph, and record the chromatogram. With the peak area (Y) as the ordinate and the ceftriaxone concentration (X) as the abscissa, the regression equation was calculated and the standard curve was drawn ( Figure 7 ).

[0158] Table 12 Ceftriaxone linearity test results

[0159]

[0160] Depend on Figure 7 As shown in Table 12, the linear equation of ceftriaxone in the range of 0.000172 mg / mL to 0.003445 mg / mL is Y=1413.5284x-0.0114, R=0.9997, the ratio of Y intercept to 100% concentration response value is 0.47%, the RSD of response factor is 7.55%, and the ratio of residual standard deviation to 100% concentration Y value is 2.11%, which meets the acceptable standard, indicating that the linearity of ceftriaxone in the range of 0.000172 mg / mL to 0.003445 mg / mL is good.

[0161] (2) Ceftriaxone Dimer D Linear

[0162] Accurately weigh 2.535 mg of ceftriaxone dimer D reference substance, dissolve and dilute to 50 ml with pH 7.0 phosphate buffer to obtain a reference substance stock solution with a ceftriaxone dimer D concentration of 0.043409 mg / ml. Accurately measure an appropriate amount of the reference substance stock solution, dilute with pH 7.0 phosphate buffer to prepare a series of ceftriaxone reference substance solutions with concentrations of 0.000347 mg / ml, 0.000434 mg / ml, 0.000868 mg / ml, 0.001736 mg / ml, 0.002605 mg / ml, and 0.003473 mg / ml, respectively. Accurately pipette 20 μl of each series of reference substance solutions, inject them into the chromatograph respectively, and record the chromatogram. With the peak area (Y) as the ordinate and the ceftriaxone dimer D concentration (X) as the abscissa, calculate the regression equation and draw the standard curve ( Figure 8 ).

[0163] Table 13 Ceftriaxone dimer D linearity test results

[0164]

[0165] From Table 13 and Figure 8 It can be seen that the linear equation of ceftriaxone dimer D in the concentration range of 0.000347 mg / mL to 0.003473 mg / mL is Y=1191.9701x-0.0960, R=0.9999, the ratio of Y intercept to 100% concentration response value is 4.95%, the RSD of the response factor is 7.85%, and the ratio of the residual standard deviation to the 100% concentration Y value is 1.13%, which meets the acceptable standard, indicating that the ceftriaxone concentration has good linearity in the range of 0.000347 mg / mL to 0.003473 mg / mL.

[0166] 2.7 Durability

[0167] (1) Solution stability

[0168] Accurately weigh 1 mg of ceftriaxone dimer D reference substance, add pH 7.0 phosphate buffer to dissolve and dilute to 25 mL as ceftriaxone dimer D stock solution. Accurately measure 1.0 mL of ceftriaxone dimer D stock solution into a 25 mL volumetric flask, add pH 7.0 phosphate buffer to dilute to the scale and shake well as ceftriaxone dimer D reference solution.

[0169] Accurately weigh 25 mg of ceftriaxone sodium for injection into a 25 mL volumetric flask, add pH 7.0 phosphate buffer to dissolve and dilute to the scale as the test solution.

[0170] Accurately measure 1.0 mL of the test solution and place it in a 100 mL volumetric flask, add pH 7.0 phosphate buffer to dilute to the mark and shake well. Accurately measure 5.0 mL of this solution and place it in a 25 mL volumetric flask, add pH 7.0 phosphate buffer to the mark and shake well to obtain the self-control solution.

[0171] After the preparation of the ceftriaxone dimer D control solution, it was placed at 2-8°C for 0, 2, 4, 8, 12, 18, 24, 40, 54, and 120 hours and at room temperature for 0, 2, 4, 8, 12, 18, and 24 hours, and 20 μL of each solution was injected into the liquid chromatograph to record the chromatogram and examine the change in the peak area of ​​the ceftriaxone dimer D.

[0172] After the test solution and the self-control solution were prepared, they were placed at 2-8°C and room temperature for 0, 3, 6, 9, 12, 18, and 24 hours, respectively. Then, 20 μL of each solution was injected into the liquid chromatograph, and the chromatograms were recorded to examine the changes in the peak area of ​​ceftriaxone dimer D in the test solution and the peak area of ​​ceftriaxone in the self-control solution.

[0173] Table 14 Ceftriaxone dimer D control solution stability test results

[0174]

[0175]

[0176] The ceftriaxone dimer D control solution showed no significant change in the main peak area of ​​ceftriaxone dimer when placed under refrigeration (5°C) for 120 hours and at room temperature for 24 hours. Therefore, it is believed that the ceftriaxone dimer D control solution is stable under refrigeration for 120 hours and stable under room temperature for 24 hours.

[0177] Table 15 Test solution stability test results

[0178]

[0179] It can be seen that there is no significant change in the peak area of ​​ceftriaxone dimer when the test solution is placed under refrigeration for 18 hours and at room temperature for 12 hours. Therefore, it is believed that the test solution is stable under refrigeration for 18 hours and stable under room temperature for 12 hours.

[0180] Table 16 Self-control solution stability test results

[0181]

[0182] As shown in Table 16, the ceftriaxone peak area of ​​the self-control solution did not change significantly within 24 hours under refrigeration and room temperature conditions. Therefore, it is believed that the self-control solution is stable within 24 hours under refrigeration and room temperature conditions.

[0183] (3) Other durability

[0184] When the ceftriaxone dimer D content of the same batch of samples was determined by changes in flow rate of ±0.1 mL / min, column temperature of ±2 °C, wavelength of ±2 nm, and pH of mobile phase A of ±0.1, the changes in the instrument's chromatographic behavior and test results were investigated to determine the durability of the method.

[0185] Table 17 Durability test conditions

[0186] Test conditions Low limit normal high limit Changes in flow rate 0.5mL / min 0.6mL / min 0.7mL / min Changes in column temperature 28℃ 30℃ 32℃ Changes in wavelength 268nm 270nm 272nm pH change of mobile phase A 4.1 4.2 4.3

[0187] ①Changes in flow rate

[0188] Chromatographic column: Sepax HP-C18, 4.6*250mm, 3μm;

[0189] Mobile phase: Mobile phase A: pH 4.2 phosphate buffer; Mobile phase B: acetonitrile;

[0190] Detection wavelength: 270nm;

[0191] Column temperature: 30°C;

[0192] Flow rate: 0.5mL / min, 0.6mL / min, 0.7mL / min.

[0193] Table 18 Experimental results at different flow rates

[0194]

[0195] ② Changes in column temperature

[0196] Chromatographic column: Sepax HP-C18, 4.6*250mm, 3μm;

[0197] Mobile phase: Mobile phase A: pH 4.2 phosphate buffer; Mobile phase B: acetonitrile;

[0198] Detection wavelength: 270nm;

[0199] Flow rate: 0.6m / Lmin;

[0200] Column temperature: 28℃, 30℃, 32℃.

[0201] Table 19 Experimental results at different column temperatures

[0202]

[0203]

[0204] ③ Changes in wavelength

[0205] Chromatographic column: Sepax HP-C18, 4.6*250mm, 3μm;

[0206] Mobile phase: Mobile phase A: pH 4.2 phosphate buffer; Mobile phase B: acetonitrile;

[0207] Flow rate: 0.6 mL / min;

[0208] Column temperature: 30°C;

[0209] Detection wavelength: 268nm, 270nm, 272nm.

[0210] Table 20 Experimental results at different wavelengths

[0211]

[0212] ④ pH changes of mobile phase A

[0213] Chromatographic column: Sepax HP-C18, 4.6*250mm, 3μm;

[0214] Flow rate: 0.6 mL / min;

[0215] Column temperature: 30°C;

[0216] Detection wavelength: 270nm;

[0217] Mobile phase: Mobile phase A: pH 4.1 phosphate buffer, pH 4.2 phosphate buffer, pH 4.3 phosphate buffer; Mobile phase B: acetonitrile.

[0218] Table 21 Experimental results of mobile phase A at different pH

[0219]

[0220] From the above experimental results, it can be seen that the polymer content was determined under different flow rates, different column temperatures, different detection wavelengths and different pH conditions of mobile phase A, and the test results met the acceptable standards, and there was no significant difference in the determination results of ceftriaxone dimer D, indicating that the method has good durability.

[0221] 2.8 Calculation of relative correction factor for ceftriaxone dimer D

[0222] According to CHP2020 version 4 Part 9102, the correction factor can be calculated from the linear slope, the formula is as follows:

[0223]

[0224] The linear equations and linear slopes of ceftriaxone and ceftriaxone dimer D and the relative correction factor of ceftriaxone dimer D are shown in the table below.

[0225] Table 23 Linear equations and slopes of ceftriaxone and ceftriaxone dimer D and relative correction factors of ceftriaxone dimer D

[0226] Compound Name Linear equations Linear slope Slope Ratio Relative correction factor Ceftriaxone Sodium Y=1413.5284x-0.0114 1413.5284 —— —— Ceftriaxone Dimer D Y=1191.9701x-0.0960 1191.9701 About 1.2 1.2

[0227] 2.9 Relative retention time of specific impurities

[0228] The relative retention times of the ceftriaxone peak and the ceftriaxone dimer D peak in the test solution were calculated under the specificity, precision and durability items, respectively. The results are shown in the table below.

[0229] Table 24 Relative retention time of ceftriaxone dimer D

[0230]

[0231] Therefore, under the detection method of ceftriaxone sodium polymer disclosed in the present invention, the relative retention time of ceftriaxone dimer D is basically stable, so the relative retention time can be used to locate ceftriaxone dimer D.

[0232] 2.10 Sample testing

[0233] The polymer content of the homemade preparation and reference preparation of ceftriaxone sodium injection was detected according to the established chromatographic conditions and test methods. The test results are shown in the table below.

[0234] Table 25 Sample content test results

[0235]

[0236] Therefore, the detection method of ceftriaxone sodium polymer disclosed in the present invention can effectively detect the polymer content in the ceftriaxone sodium injection sample, and there is no obvious difference in the polymer content between the homemade preparation and the reference preparation.

[0237] The above description of the disclosed embodiments enables one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for detecting a ceftriaxone sodium polymer, characterized in that: The C18 reversed phase high performance liquid chromatography method is used to detect the ceftriaxone sodium polymer that needs to be controlled. The chemical name of the ceftriaxone sodium polymer is: (6R,7R)-7-((Z)-2-(2-aminothiazol-4-yl)-2-(methoxyimino)acetylamino)-3-(((4-((Z)-2-(((6R,7R)-2-carboxyl-3-(((2-methyl-5,6-dioxo- -1,2,5,6-tetrahydro-1,2,4-triazine-3-yl)thio]methyl)-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-en-7-yl)amino)-1-(methoxyimino)-2-oxoethyl)thiazol-2-yl)amino)methyl)-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid; the structural formula is:

2. The method for detecting ceftriaxone sodium polymer according to claim 1, characterized in that: The chromatographic conditions of the C18 reverse HPLC method are: Octadecyl bonded silica was used as the filler; the column temperature was 28-32°C, the flow rate was 0.5-0.7 mL / min, and the detection wavelength was 268-272 nm; 0.05 mol / L phosphate buffer was used as mobile phase A, and acetonitrile was used as mobile phase B; The elution procedure is shown in Table 1: Table 1 3. The method for detecting ceftriaxone sodium polymer according to claim 2, characterized in that: The octadecyl bonded silica gel is filled with C18, with a particle size of 3 μm, 4.6×250 mm, or a chromatographic column with equivalent performance; The preparation method of the 0.05 mol / L phosphate buffer is as follows: 9.0 g of disodium hydrogen phosphate dodecahydrate and 3.5 g of anhydrous potassium dihydrogen phosphate are taken, dissolved in water and diluted to 1000 mL, and the pH is adjusted to 4.1-4.3 with phosphoric acid.

4. The method for detecting ceftriaxone sodium polymer according to claim 2, characterized in that: The detection method of the C18 reverse HPLC method is: 1) Take an appropriate amount of ceftriaxone sodium and obtain a system adaptability solution through pretreatment; take 20 μL of this solution and inject it into the chromatograph, record the chromatogram, and the separation degree between the ceftriaxone peak and the ceftriaxone sodium polymer peak and its previous adjacent peak should meet the requirements; 2) Take an appropriate amount of ceftriaxone reference substance, add pH 7.0 phosphate buffer to dissolve and quantitatively dilute to make a 0.2 μg / mL solution, and shake well as the sensitivity solution; Take 20 μL of this solution and inject it into the chromatograph, record the chromatogram, and the signal-to-noise ratio of the ceftriaxone peak should be no less than 10; 3) Take an appropriate amount of ceftriaxone sodium, dissolve it in pH 7.0 phosphate buffer and quantitatively dilute it to make a 1 mg / mL solution, and shake well as the test solution; Take 20 μL of this solution and inject it into the chromatograph, record the chromatogram; accurately measure an appropriate amount of the test solution and add pH 7.0 phosphate buffer to dilute it to make a 2 μg / ml solution, shake well as the control solution; Take 20 μL of this solution and inject it into the chromatograph to record the chromatogram.

5. The method for detecting ceftriaxone sodium polymer according to claim 4, characterized in that: The relative retention time of the ceftriaxone sodium polymer is about 1.21, and the relative correction factor is about 1.

2.

6. The method for detecting ceftriaxone sodium polymer according to claim 4, characterized in that: The preparation method of the phosphate buffer of pH 7.0 is: Take 6.805 g of anhydrous potassium dihydrogen phosphate and 0.94 g of sodium hydroxide, weigh accurately, add appropriate amount of water to dissolve, dilute with water to 1000 mL, adjust the pH value to 7.0 with 1 mol / L sodium hydroxide solution, and shake well to prepare a pH 7.0 phosphate buffer.

7. The method for detecting the ceftriaxone sodium polymer according to claim 4, characterized in that: The pretreatment method for obtaining the system suitability solution through pretreatment in step 1) is: Take an appropriate amount of ceftriaxone sodium, add pH 7.0 phosphate buffer to dissolve and dilute to a 5 mg / mL solution; accurately measure 1 mL of this solution, add 0.8-1.5 mL of 0.1 mol / L hydrochloric acid to destroy it for 0.5-1.2 hours, then add 0.1 mol / L sodium hydroxide solution to neutralize it, and add pH 7.0 phosphate buffer to dilute to a 0.25 mg / mL solution as the system adaptability solution; Alternatively, take an appropriate amount of ceftriaxone sodium, add pH 7.0 phosphate buffer to dissolve and dilute to a 50 mg / mL solution; accurately measure 1 mL of this solution, place it in a transparent glass container, and then continuously destroy it under a light intensity of 5000±500Lx for 4±1 hours, and then add pH 7.0 phosphate buffer to dilute it to a 0.25 mg / mL solution as the system adaptability solution.

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